1. Field of the Invention
The present invention is in the area of thermostable enzymes, and the use of same. Especially, the invention is in the area of Chaetomium xylanases that are active at a high temperature. The compositions of the invention are useful to modify plant biomass properties. The invention is also directed to a method for enzyme-aided bleaching using the enzyme compositions of the invention. Furthermore, the invention concerns the use of the novel enzyme compositions in feedstuffs and flour compositions.
2. Description of Related art
Plant biomass is a composite material consisting primarily of a matrix of cellulose, hemicellulose, and lignin. Enzymes degrading e.g. the hemicellulose xylan, xylanases, can be used in animal feed compositions which are rich in arabinoxylans and glucoxylans, in baking, and for bleaching of cellulosic pulps.
Thus, when added to feeds for monogastric animals (e.g. poultry or swine) which contain cereals (e.g. barley, wheat, maize, rye or oats) or cereal by-products, a hemicellulolytic enzyme improves the break-down of plant cell walls which leads to better utilization of the plant nutrients by animal. This leads to improved growth rate and feed conversion. Also, the viscosity of the feeds containing xylan can be reduced.
In baking applications small amounts of xylanases added to the flour impart favorable characteristics to the dough and to the bread itself. Such characteristics include e.g. increased loaf volume and better textural characteristics (break and shred quality and crumb quality).
In the pulp and paper industry xylanases and other hemicellulases are used, e.g., to improve the bleachability of the pulp.
The aim of kraft pulp bleaching is to remove the residual lignin that is left in pulp after kraft cooking. Traditionally, this has been done using chlorine-containing chemicals. Because of environmental concerns and consumer demands, alternative bleaching technologies have been desired.
The first biotechnical approach to this problem was to attack the lignin directly with lignin degrading enzymes. However, the chemistry of enzymatic lignin degradation seems to be very complicated and difficult to control.
Lignin can be degraded, if the whole microorganism that produces ligninases is used. However, treatment times are relatively long. For example, treatment times may take days, and the microorganisms need supplemental nutrients to work. It can also be difficult to control the growth of other, undesired, microbes. Lignin degradation by using ligninases or by microorganisms is the subject of much research. (see, for example, Farrell, R. L. et al., Lignocellulosics 305-315 (1992); Jurasek, L., Lignocellulosics 317-325 (1992)).
In addition to cellulose and lignin, wood pulp contains hemicellulose. Another approach to lignin removal is to attack hemicellulose--the third main component of wood. The hemicellulose in native hardwood is mainly xylan, while in softwood the hemicellulose is mainly glucomannans and some xylan. During kraft cooking, part of the xylan is dissolved into the cooking liquor. Towards the end of the cooking period when the alkali concentration decreases, part of the dissolved and modified xylan reprecipitates back onto the cellulose fiber.
In 1986, it was noticed that xylanase pretreatment of unbleached kraft pulp results in a lessened need for chemicals in the bleaching process (Viikari, L. et al., Proceedings of the 3rd Int. Conf. on Biotechnology in the Pulp Paper Ind., Stockholm (1986), pp. 67-69). Xylanase pretreatment of kraft pulp partially hydrolyses the xylan in kraft pulp. This makes the pulp structure more porous and enables more efficient removal of lignin fragments in the subsequent bleaching and extraction stages. Later, in several laboratories, the xylanase pretreatment was reported to be useful in conjunction with bleaching sequences consisting of Cl.sub.2, ClO.sub.2, H.sub.2 O.sub.2, O.sub.2 and O.sub.3. See reviews in Viikari, L. et al., FEMS Microbiol. Rev. 13: 335-350 (1994); Viikari, L. et al., in: Saddler, J. N., ed., Bioconversion of Forest and Agricultural Plant Residues, C-A-B International (1993), pp. 131-182; Grant, R., Pulp and Paper Int. (September 1993), pp. 56-57; Senior & Hamilton, J. Pulp & Paper: 111-114 (September 1992); Bajpai & Bajpai, Process Biochem. 27:319-325 (1992); Onysko, A., Biotech. Adv. 11:179-198 (1993); and Viikari, L. et al., J. Paper and Timber 73:384-389 (1991).
As a direct result of the better bleachability of the pulp after such a xylanase treatment, there is a reduction of the subsequent consumption of bleaching chemicals, which when chloride containing chemicals are used, leads to a reduced formation of environmentally undesired organo-chlorine compounds. Also as a direct result of the better bleachability of pulp after a xylanase treatment, it is possible to produce a product with a high final brightness where such brightness would otherwise be hard to achieve (such as totally chlorine free (TCF) bleaching using peroxide). Because of the substrate specificity of the xylanase enzyme, cellulose fibers are not harmed and the strength properties of the product are well within acceptable limits.
However, in many of the practical applications, the use of xylanases is not straightforward; the xylanases must be active in the temperature and pH conditions of the process in which they are used. Formulation of commercial feed using pelleting, extrusion or expanding, often contains steps involving high temperatures (70-180.degree. C.). Enzymes added to the formulation process should withstand these conditions. On the other hand, the corresponding temperature in the intestin of animals is about 40.degree. C. Thus, ideal xylanases for feed compositions should withstand the above mentioned extream temperatures. In bleaching applications, xylanase application is not as simple as adding a xylanase treatment step. Because the bleaching process, and even the sequence of the steps used in the bleaching process varies in different pulp mills, there is thus a continous need to find new xylanases active in different temperature and pH conditions.
Most commercial xylanases designed for feed applications and pulp bleaching are not very thermo-tolerant, especially when neutral or alkaline pH conditions are used. In practice, xylanases are generally inefficient or inactive at temperatures higher than 60.degree. C. and often these enzymes work under acidic conditions. Generally, there are differences in the physical characteristics of xylanases of fungi and bacteria (for review, see Wong et al., Microbiol. Rev. 52:305-317 (1988)). Typically, fungal xylanases have temperature optimum at about 50.degree. C. and lower pH optimum than have those of bacterial origin. Xylanases of bacterial origin generally have a temperature optimum in the range of 50 to 70.degree. C.
PCT/US90/05933 (WO 91/05908) proposes the use of xylanase in pulp bleaching together with chlorine or chlorine compounds. Chaetomium is proposed as a xylanase source. Screening for xylanase from Streptomyces and Chainia strains is described. Bleaching experiments were performed using xylanase preparations from Chainia sp. culture medium.
EP-A 0 406 617 proposes the use of xylanase in an enzymatic delignifying process of lignocellulosic material, especially after a ligninolytic enzyme. The xylanase may be derived form various sources, for example from Chaetomium. The use of xylanase from Chainia sp culture medium is exemplified.
Gandhi, J. P. et al., J. Chem. Tech. Biotechnol. 60:55-60 (1994) reported studies on thermostability and pH stability of crude xylanase preparations from Chaetomium globosum. The optimum temperature of the xylanase was found to be in the range of 50 to 60.degree. C., while the optimum pH was found to be pH 5.0. The enzyme was reported not to lose any of the original activity between 40 to 60.degree. C. for a period of 10 min and was reported to retain more than 70% of the original activity in the range of 70 to 100.degree. C. for 10 min. The pH stability studies indicated that the enzyme retained all activity between pH 5 and 6 and more than 70% of the original activity over a wide range of alkaline pH values (7-10). It was suggested to use the culture filtrates for the treatment of cellulose pulps without further purification.
The xylanase of Chaetomium cellulolyticum and Chaetomium trilaterale have also been studied (Dubeau, H. et al., Biotechnol. Lett. 9:275-280 (1987) and Kawaminami, T. and Iitzuka, H. J. Ferment. Technol. 48:161-168 (1970), respectively). However, neither the thermostability nor the pH profile of the enzymes was reported. The xylanases were suggested to be of use in clarification of fruit juices. The use of these enzymes in pulp bleaching or as feed additive was not suggested.
Ganju, R. K. et al., Can. J. Microbiol. 35:836-842 (1989) reported the purification and characterization of two xylanases from Chaetomium thermophile var. coprophile. Two xylanases (I and II) out of several extracellular xylanases produced by C. thermophile var. coprophile were purified to homogeneity. These enzymes had molecular weights of 26,000 Daltons (xylanase I) and 7,000 Daltons (xylanase II). The temperature optima for xylanase I and II were 70 and 60.degree. C., and they were optimally active at pH 4.8-6.4 and 5.4-6.9, respectively. The use of these xylanases in pulp bleaching or as feed additive was not suggested.
Irie et al., Hakko Kogaku Kaishi 70(2): 109-114 (1992) have reported the purification of a xylanase from a Chaetomium gracile mutant. The molecular weight was reported to be 19,000 daltons, and the xylanase contained two subunits: one having a molecular weight of 14,400 daltons and the other a molecular weight of 4,800 daltons. The pI was 8.35. The maximal xylobiose forming activity was found at pH 5.0 and 50.degree. C. The pH range was reported to be pH 4.0-pH 7.0. Yoshino et al., Curr. Genet. 29:73-80 (1995) reported the isolation and sequencing of two xylanase genes from Chaetomium gracile wild and mutant strains and their expression in Aspergillus nidulans. The mature CgXA and CgXB xylanases contain 189 and 211 amino acids, respectively, and share 68.5% homology. The cgxA and cgxB genes were introduced into Aspergillus nidulans and reported to be expressed with their own promoters. The use of these C. gracile xylanases in pulp bleaching or as feed additive was not suggested.